Gonadotropins are a family of hormones, which are essentially mainly involved in the fertility cycle in females and males. Gonadotropins can be derived from urine, both for research and treatment purposes, however several gonadotropins can be produced recombinantly.
In particular, gonadotropins can be employed in the treatment of infertility.
The four main gonadotropins which are involved here and which all belong to the same glycoprotein family are follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH) and chorionic gonadotropin (hCG). All of these gonadotropins consist of an alpha and a beta subunit; the alpha subunit is common to all, i.e. the same for all above-mentioned four gonadotropins, while the beta subunit differs, respectively.
As mentioned above, the gonadotropins are a group of heterodimeric glycoprotein hormones which regulate gonadal function in the male and female. They include follicle stimulating hormone (FSH), luteinising hormone (LH), thyroid stimulating hormone (TSH) and (human) chorionic gonadotropin (hCG).
FSH is naturally secreted by the anterior pituitary gland and functions to support follicular development and ovulation. FSH comprises a 92 amino acid alpha subunit, also common to the other glycoprotein hormones, e.g. LH and hCG, and a 111 amino acid beta subunit unique to FSH that confers the biological specificity of the hormone (Pierce and Parsons, 1981, Glycoprotein hormones: structure and function, Ann Rev Biochem., 50: 465-495). Each subunit is post-translationally modified by the addition of complex carbohydrate residues. Both subunits carry two sites for N-linked glycan attachment, the alpha subunit at amino acids 52 and 78 and the beta subunit at amino acid residues 7 and 24 (Rathnam and Saxena, (1975) Primary amino acid sequence of follicle stimulating hormone from human pituitary glands. I. alpha subunit, J Biol Chem. 250 (17):6735-6746; Saxena and Rathnam, (1976) Amino acid sequence of the beta subunit of follicle-stimulating hormone from human pituitary glands, J Biol Chem. 251(4): 993-1005)). FSH is thus glycosylated to about 30% by mass (Dias and Van Roey, (2001) Structural biology of human follitropin and its receptor. Arch Med Res. 32(6): 510-519; Fox et al. (2001) Three-dimensional structure of human follicle-stimulating hormone. Mol Endocrinol. 15(3), 379-89).
FSH purified from post-menopausal human urine has been used for many years in infertility treatment; both to promote ovulation in natural reproduction and to provide oocytes for assisted reproduction technologies. Two recombinant versions of FSH, Gonal-f (Merck Serono) and Puregon (Schering-Plough) became available in the mid-1990s. These are both expressed in Chinese hamster ovary (CHO) cells (Howles, C. M. (1996), genetic engineering of human FSH (Gonal-f), Hum Reprod. Update, 2: 172-191). CO is frequently used in infertility treatments because of this compound having an LH activity.
Both human FSH and hCG are heterodimers composed of an alpha and a beta subunit. The alpha subunit in both hormones is identical. Differences between the two hormones are conferred by the beta subunit. The mature beta subunit of FSH is composed of 111 amino acids, while that of hCG is composed of 145 amino acids, additionally the primary amino acid sequence of the FSH and hCG beta subunit are differing throughout the whole beta chain. The beta chain of both FSH and hCG contains six disulfide bridges, due to their different amino acid sequence they do however differ in their higher order structure, resulting in a different folding and distribution of charged, polar and hydrophobic regions (Fox et al. (2001) Three-dimensional structure of human follicle-stimulating hormone. Mol Endocrinol. 15(3), 379-89).
Although both beta subunits of FSH and hCG are glycosylated, the beta subunit of FSH contains only N-glycosylation (N-7 and N-24) while the beta subunit of hCG contains both N- and O-glycosylation (N-13, N-30, O-121, O-127, O-132 and O-138). The extra glycosylation in the beta subunit of hCG makes it more hydrophilic than that of FSH. β-subunits provide specificity for the receptor interaction.
CHO cells are commonly used for the production of pharmaceutical recombinant proteins. Structural analysis has identified that sialic acid is exclusively attached by an α2,3-linkage. Many human glycoproteins contain a mixture of both α2,3- and α2,6-linkages for sialic acid residues. Therefore, recombinant proteins expressed using the CHO system will differ from their natural counterparts in their type of terminal sialic acid linkages.
Infertility
In the present context, “infertility” shall be defined as the diminished ability or the inability to conceive and have offspring. Women who are able to get pregnant but then have repeat miscarriages are also said to be infertile. Infertility is also defined in specific terms as the failure to conceive after a year of regular intercourse without contraception. Infertility can be due to many causes. Studies have shown that a little more than half of cases of infertility are a result of female conditions. The remainder are caused by sperm disorders and by unexplained factors. There are currently several possibilities to treat infertility.
Those are a timed intercourse, the use of assisted reproductive technologies (ARTs), a medical management of endometriosis, fibroids and female sexual dysfunction (FSD), and surgery to correct abnormalities. In assisted reproductive technology, drugs to stimulate ovulation are used. Next to LH and hCG, FSH is one of those compounds which is used in this context.
For administration, liquid formulations of these compounds are suitable. Unfortunately, it has been shown in the past that the preservatives added to liquid formulations, in particular benzyl alcohol (BA), phenol and m-cresol, exert a destabilizing effect on the protein (Maa, Y. F. and Chung, C. H. 1996, Aggregation of recombinant human growth hormone induced by phenolic compounds. Int. J. Pharm. 140:155-168; Lam, X. M., Patapoff, T. W., and Nguyen, T. H. 1997, The effect of benzyl alcohol on recombinant human interferon-gamma. Pharm. Res. 14:725-729; Hoffmann, J. A. and Lu, J. 2002, FSH and FSH variant formulations comprising benzyl alcohol as a preservative. EP0974359B1, 1-50).
It is therefore important to provide a stabilized formulation, in particular in view of the fact that the dosage of the FSH to be administered should reduce the risk of side effects of dissociated or aggregated forms like immunogenic responses, if non-native FSH is present. The destabilizing events resulting from preservatives however decrease the actual level of active gonadotropin, i.e. FSH, within a liquid formulation. Thus, it is an aim of the present invention to provide formulations, in particular liquid formulations of FSH, which are stable, as well as a method for their stabilization.